FIGURE 4.

Restoration of G₁ gene expression and S phase entry upon Brd4 re-expression. A, NIH3T3 cells were transduced with control or Brd4 rescue vector plus Brd4 shRNA vector (Ctrl, Brd4 KD, and Rescue). Cells were synchronized by serum starvation, released, and incubated for 12 h. Expression of indicated proteins was analyzed by immunoblot. pRb, phosphorylated Rb. B, cells transduced as above (Ctrl, Brd4 KD, Rescue) were synchronized and allowed to proceed through G₁ for the indicated times (h) and analyzed for expression of indicated genes by qRT-PCR. Values were normalized to 18 S rRNA. C, cells (Ctrl, Brd4 KD, Rescue) were allowed to exponentially grow for 14 days, and total cell yields were estimated as in Fig. 1B. D, Ctrl, Brd4 KD, and Rescue cells were synchronized as above, and cell cycle profiles were examined by flow cytometry as in Fig. 1C. See the quantification in supplemental Fig. S1D.